Design and synthesis of opioid peptide ligands. Much of our research emphasizes the design and synthesis of conformationally restricted analogs of native, receptor active peptides (especially opioid peptides) as an approach to compounds with higher receptor and pharmacological selectivity. The rationale for the approach is that less flexible peptides may exhibit preferred interactions with one of a set of receptor subtypes leading to increased selectivity. The conformational analyses of these conformationally restricted ligands utilize both experimental (NMR, xray) and theoretical approaches (conformational search /molecular mechanics) to arrive at a pharmacophore model. The model so generated is then employed as a template for the design of new analogs aimed at testing the conformational model and as improved (more selective and/or more potent) compounds.
Design and synthesis of peptidomimetics. Peptides, in general, have two liabilities that make them undesirable candidates for drugs. First, they are typically enzymatically labile and, second, they are often too large for good bioavailability. To circumvent these liabilities we are pursuing the design and synthesis of nonpeptide structures which maintain the key elements required for activity but which replace the labile peptide bonds with more stable features and which are stripped of unessential structural components.
Modeling of opioid receptors and other GPCRs. To obtain  atomic resolution structures of the transmembrane 7-a -bundle of opioid receptors, we have developed a theoretical approach, based on the analysis of correlations in multisequence alignments of 410 GPCRs  to pack the 7 a-helices using the distance geometry algorithm with an evolving system of interhelical H-bonds, formed by polar side chains in various proteins within the family and collectively applied as distance constraints [Pogozheva et al., 1997]. This approach has been used to calculate  models of the transmembrane 7 a -bundle of 30 different GPCRs and receptor-like proteins.
NMR studies of synthetic peptides derived from extracellular loops.   Structural information about extracellular loops of GPCRs is practically absent. However, in many receptors these loops are important for binding of large ligands.   For example, large opioid peptide ligands interact with both transmembrane and extracellular regions of the receptors. Consequently we are pursuing the synthesis of linear and cyclic peptides, derived from the extracellular domain of opioid receptors, and the conformational analysis of these peptides by 1H NMR. The synthetic peptides, corresponding to extracellular loops 1, 2, and 3 with small fragments of adjacent helices will be studied separately and in complexes stabilized by disulfide bonds and through the binding of metal ions to artificially designed His-clusters . The structural information obtained will be used for the refinement of the preliminary calculated structures of the extracellular loops.
Protein engineering of GPCRs includes the design of mutants and chimera of opioid receptors (ORs) and  muscarinic acetylcholine receptors (MRs) in collaboration with H. Akil and H. LeVine, respectively. Metal-ion-binding centers, formed by His and Cys residues have been designed in ORs between adjacent transmembrane helices, between helices and extracellular loops, and between different extracellular loops, based on the calculated structures of the transmembrane domain of ORs and preliminary structures of their extracellular loops.  Chimeric MRs are built from the cloned six-a -bundle and synthetic seventh transmembrane helix. Reconstitution of a  complete functional receptor from these fragments allows helix packing interactions within the transmembrane a -bundle to be examined.
Synthesis and studies of nitric oxide synthase-derived calmodulin-binding peptides.  The activity of the constitutive and inducible isoforms of nitric oxide synthase (nNOS and iNOS) both require the binding of calmodulin, however nNOS requires calcium to bind calmodulin, whereas iNOS co-purifies as a calcium/calmodulin complex. These results suggest different and unique aspects of calmodulin recognition.  It has been shown that peptides corresponding to the calmodulin binding region of iNOS (iP) and nNOS (nP) completely inhibit their corresponding enzyme isoforms, however, nP is unable to inhibit iNOS, consistent with the conclusion that that calmodulin interacts with the two isoforms of NOS differently. We are investigating this possibility through the synthesis and evaluation of enzymatic inhibitory potency of analogs of iP and through the study, via NMR, of interactions between these peptides and calmodulin.
Development of the theory of protein structure is an important step toward the prediction of the structure of differents proteins, including the calculation of entire  structure (with extramembrane domains) of GPCRs and for the docking of peptide ligands to the receptors. The goal of this project is to develop a quantitative theory that describes formation, growth, adjustment, and association of a -helices and b sheets during protein folding, and finally to predict the 3D structure of proteins.

 This page was last updated 05/01/98 by I. D. Pogozheva